Posts from Behind the Bench @ Life Technologies

How to deliver absolutely attainable digital PCR, simply and affordably

This is the second in a six part series to introduce you to digital PCR (dPCR). In this edition, we’ll take a look at how QuantStudio® 3D dPCR from Life Technologies offers simplicity, affordability, and most importantly, absolute quantification attainability.

At the Advances in Genome Biology and Technology, there were a wide variety of applications from the scientific posters that illustrate the flexibility of sequencing to solve a variety of problems. MRI Global is a not-for-profit contract research organization, and Dr. Jonathan Jacobs, (or as he is known on Twitter as @bioinformer) research is centered on air sample for biosurveillance and how pathogens may impact public health using the Ion Proton™ Sequencing System.

The rapidly-growing field of pharmacogenomics (PGx) is the study of genetic variation as it relates to drug response, determined by variation in drug metabolism enzyme and transporter (DMET) genes. The significance of PGx will increase as personalized medicine becomes more widely adopted.

In 1951, Fred Sanger first determined the sequence of two proteins (the bovine insulin A and B forms), showing that they were distinct molecules, and for this he was awarded a Nobel Prize in Chemistry in 1958. In 1965, he lost the race to be the first to sequence a tRNA molecule. In 1977, he introduced the “di-deoxy” chain-termination method, which earned him a second Nobel Prize in Chemistry in 1980 (which he shared with Paul Berg and Walter Gilbert, of the Maxam-Gilbert chemical-based method of sequencing ).

Using a panel of 740 microRNAs to research testicular germ-cell seminomas, Dr. Martin Rijlaarsdam of Erasmus University Medical Center in the Netherlands describes in this LabChat their characterization of unique biomarkers.

To date, qPCR has been a powerful and sensitive gene analysis techniques used for a broad range of applications. As the name suggests, qPCR measures PCR amplification as it occurs, unlike traditional PCR, which collects results after the reaction is complete, making it impossible to determine the starting concentration of nucleic acid.

At the Advances in Genome Biology and Technology conference we interviewed a close collaborator of his, Dr. Andrea Kohn also at the University of Florida, who gave a presentation entitled “Single-Cell Methylome Profiling: Integration of a Transcriptome and Methylome From Individual Identified Neurons”.

Bobby Sebra of the Icahn School of Medicine at the Mount Sinai University Hospital discusses the development of a 25,000-plex Ion AmpliSeq™ Panel as part of the Resilience Project, at the recent Advances in Genome Biology and Technology conference.

We’ve talked about the Resilience Project before on Behind the Bench, and Dr. Sebra talks about the technical underpinnings and performance of their large AmpliSeq™ Panel.

David Guttery of the University of Leicester UK spoke with us at the Advances in Genome Biology and Technology meeting, and shared his experience in developing an Ion AmpliSeq™ Panel for targeted sequencing of tumor-related genes for cancer research.

At the Advances for Genome Biology and Technology, Dr. Jason Johnson of Thermo Fisher Scientific presents a new capability to examine circulating free DNA (cfDNA), circulating tumor cells (CTC) and germline DNA variants from the same sample.

And from there he discusses Ion AmpliSeq™ panel analysis with sensitive minor allele frequency detection.

Claritas Genomics CEO Dr. Patrice Milos shares the highlights of her presentation along with feedback about the new Ion Hi-Q™ chemistry in this interview from the Advances in Genome Biology and Technology. Their laboratory automation and flexible sequencing service as an Ion Torrent™ Certified Service Provider offer fast turnaround time with the ability to scale from smaller to larger whole-exome sequencing projects.

Currently there is a lot of interest in circulating tumor cells (CTC’s) as a rich source of biomarkers for the early cancer detection. For these enrichment methods, the detection of cancerous cells is clouded by the presence of many normal cells. Thus there is a need for sensitive somatic mutation detection.

Mate-pair libraries for microbial whole genome sequencing now available

Earlier this year, a new microbe dubbed a “methanogen” was reported in the now-thawing permafrost of northern Sweden as part of a study. This microbe appears to play a significant role in methane release due to global warming. Around the same time, a Russian team’s study revealed new insights into the genetic changes undergone by Mycobacterium tuberculosis, which may help researchers understand how the tuberculosis-causing microorganism can achieve drug resistance.

In this video interview she discusses differential gene correlations to two different orthogonal gene expression measurements: a panel of over seven hundred TaqMan® genes, and whole-transcriptome RNA-Seq.

During the Advances in Genome Biology and Technology Conference Dr. Mark Andersen of Thermo Fisher R&D shared highlights of his presentation entitled “You want to put what where? Ion AmpliSeq™ technology, automation, any sample”. If you want to see what else the Ion Chef™ instrument can do, take a look!

At the Advances in Genome Biology and Technology Conference Dr. Adam Harris of Thermo Fisher Scientific R&D shared highlights of a presentation called “An avalanche of data: long reads and a speedy workflow with isothermal amplification”. How much time does this process save in template preparation? Watch and find out!

Here at Advances in Genome Biology and Technology there was a software bioinformatics demonstration session; we spoke with Dr. Carl Dowds Sr. Product Manager of Informatics Applications about the latest version of Ion Reporter™ Software and what it offers.

Studying genetic variation generally lays aside consideration for variants as they line up along a particular parental allele. While we accept that the human genome is diploid, in practice having two different forms of each allele is taken as individual homozygous or heterozygous single nucleotide variants at a given base position, rather than a string of variants lined up along distinct haploid genes.

Five years ago, Dr. Richard Gibbs of the Baylor College of Medicine gave a memorable plenary presentation at the annual Advances in Genome Biology and Technology conference in Marco Island, Florida."

Entitled “Genome Sequencing to Health and Biological Insight”, in his introduction he illustrated the seesaw battle between whole-genome or whole-exome sequencing approaches to a targeted sequencing approach by showing a drawing of two aluminum cans of popular cola soft-drinks, as a cartoon with boxing gloves. Illustrating this question of approaches as a battle, he said that while he favored the whole-genome / whole-exome sequencing approach he didn’t see any end to this struggle for the foreseeable future.

From clinical research, agricultural biotechnology, metagenomics, and technical improvements, here's a brief video roundup from yourteam at Behind the Bench of the major next-generation sequencing "moments that mattered" in 2014.

What would you do with free transcriptome sequencing? We asked that question in November 2014 during the Ion Torrent™ Transcriptome Profiling Grant Program. And over 200 researchers world-wide answered by submitting grant proposals for up to 24 samples of free sequencing using the Ion AmpliSeq™ Transcriptome Human Gene Expression Kit, with sequencing services provided by our Certified Service Providers. Here are the winners...

While testicular cancer is relatively rare (about 8,500 cases in the US annually) it accounts for the most common solid tumor type in men aged 15 to 35. The vast majority (95%) are germ cell tumors, of which about half are called seminomas. These are usually slow growing and when discovered early are often treatable. The other half are called nonseminomas, are often more aggressive and can metastasize to other locations in the body.

Silicon Biosystems is a new company based in Italy that is developing a novel platform for solving the problem of heterogeneity of cells from tissues of many types. (For example, it is well-established that cancer is comprised of at least six cell types.) Their system uses microfabricated electrodes on a silicon chip married to a fluidic system, where a set of 9 electrodes form a di-electrophoretic ‘cage’ that enables single-cell trapping (as well as manipulation). These chips are called a DEPArray.

Nitrogen availability is one of the major limiting factors to crop growth. In the developed world where the use of nitrogen fertilizers are ubiquitous, their over-use causes nitrification of the environment and damaging algal blooms, in addition to atmospheric pollution. In the developing world, little access to fertilizers by smallholder farmers limits crop production, much more than the availability of water.

At the Plant and Animal Genome conference (held annually in San Diego California), many specialized sessions are devoted to specific crops and livestock. For example, on one afternoon there were 11 concurrent sessions, among which were Coffee Genomics, Curcurbit (otherwise known as pumpkins, squashes and gourds), and Domestication Genomics.

Circulating Tumor Cells (CTC) have rightly attracted a lot of interest in the field of cancer research, and in this interview Dr. Mitch Garcia discusses work that comes out of Dr. Hsian-Rong Tseng’s group at the University of California, Los Angeles.

new cases in the United States in 2014 alone. About 5% of these cases are inherited in an autosomal dominant fashion, and the two major types of these inherited cases are called familial adenomatous polyposis and hereditary non-polyposis colorectal cancer (HNPCC).

King Richard III had a short but notable reign (1483-1485), starting with the death of his brother, his 12-year old nephew Edward V declared an illegitimate heir to the throne, then Richard III ascending to the throne. His tumultuous reign ended when he was killed in battle by the forces of Henry VII. At the age of 32, he was the last of English Royalty to die in battle, and the only king whose remains were not known.

Kristian Ridley from Thermo Fisher Scientific shares what is new about genetic analysis from the exhibit floor of the recent Plant and Animal Genome Conference, along with observations about which products Thermo Fisher customers are interested in.

Speaking at the Plant and Animal Genome Conference, Dr. Jason Boone discusses Floragenics’ work on a variety of animal genomes in varied locales worldwide, as a genomics service provider. They have used genetics to track the pygmy elephant for conservation efforts, and also discusses the use of Ion AmpliSeq™ to perform rapid genotyping-by-sequencing with barley, and the cross-platform accuracy they’ve experimentally determined.

Don’t be paralyzed by too many choices for custom gene expression and genotyping assays

Having freedom to choose is a great thing. Having too many choices to choose from can be paralyzing. We have all been at a restaurant where the extensive menu meant that not only did it take a long time to make up your mind on what to have for dinner, but also it generated some concern that the optimal choice was not made. And then once the meal arrives for everyone at the table, this concern can likely turn into regret.

GeneSeek a genetic service provider, has been characterizing traits for agricultural biotechnology companies since 1998, with many capabilities across the genetic analysis spectrum including genotyping and SNP microarray technologies.

Cassava is a vitally important food crop worldwide. Otherwise known as manioc; in North America a well-known derivative form is tapioca. Native to northern Brazil, it spread throughout the South American continent and was carried by the Spanish and Portuguese explorers to the West Indies, then to the continents of Africa and Asia. Cultivated worldwide, an estimated 800 million people depend on it (500 million in Africa) as a main source of calories in their diet (up to 60%).

At the recent Plant and Animal Genome Conference, Monsanto computational biologist Dr. Lex Flagel discusses their work on the Western Corn Rootworm and the use of genotyping-by-sequencing to understand the genetic mechanism to Bt toxin resistance.

The International Rice Research Institute, located in Los Baños, Philippines, aims to reduce poverty and hunger, improve the health of rice farmers and consumers, and ensure environmental sustainability of rice farming.

James Schnable from the University of Nebraska-Lincoln has developed a technology called ‘tunable Genotyping by Sequencing’ (tGBS) that provides much better read-depth compared to conventional genotyping-by-sequencing approaches.

We are living in a remarkable, connected age – where all the world’s information is just a click away. It is hard to imagine that as few as twenty years ago to look up a publication abstract it meant going to an academic library and pull out the ISI Abstracts

reference volume(s). It seems quaint now, as looking up abstracts of journal articles are just a few clicks away.

You may be familiar with the Ion AmpliSeq™ Panels for highly multiplexed, single-tube PCR for next-generation sequencing target selection for human or mouse genes. You also may be familiar with the online AmpliSeq.com tool for making custom designs, as well as accessing gene lists and target coordinates for the standard research panels that are offered.

But did you realize that there are now several additional reference genomes to design a custom research panel from?

Dan Tonge has been working with the Ion Torrent™ platform for some time, and currently is working with uncultured fungi at very low concentrations. He discusses his approach toward how these amplicons are designed, and how they tested and challenged their ability to distinguish between closely related fungal species.

A huge neuroscience project is just getting started: will you be a part of it?

When you think of the largest government undertakings that had the largest impact in the modern era, what comes to mind? (By ‘impact’ you can consider the economic, social, or scientific ramifications excluding war, which of course is a separate question.)

A few talks and a workshop of interest at PAG XXIII January 10-14, 2015

The Plant and Animal Genome Conference calls itself ‘the largest Ag-Genomics meeting in the world’, and there is no reason to contest that. Do you work with arthropods like psyllids? There’s something for you. Aquaculture? There’s a day-long program devoted to it. Cacao? A whole afternoon set aside, now of particular importance given recent publicity around the growing worldwide chocolate deficit.

We’ve all grown accustomed to turning to YouTube to answer questions ranging from child care to yoga tips, or even how to deep fry a turkey. Back in 2012, we launched the Ask TaqMan video series with the belief that researchers are hungry for knowledge on a topic that’s very near and dear to our hearts: qPCR.

As it turns out, A LOT of people are in need of qPCR help, and in 2014 alone, the Ask TaqMan videos were viewed over 120,000 times! So what were those burning qPCR questions in 2014? Let’s take a look at the top 5 for the year…

Joel Dudley of the Icahn School of Medicine, The Mount Sinai Hospital (New York New York USA) discusses big data in the age of genomics. As a data scientist involved with the Resilience Project and its data-driven approach toward new discoveries with major research implications.